Structure and dynamics of transcriptionally active chromatin domains of Drosophila melanogaster interphase chromosomes

我们目前的数据表明，唾液腺中的61C7-8基因是由一个~20kb的开放染色质结构域组成的，该结构域包含四个蛋白质编码基因和一个非编码转录本。通过定点定向重组，我们在相同的异位位置产生了野生型和部分缺失的基因座，这是最初用细胞遗传学方法表征的。在本项目中，我们想要研究高分辨率形成61C7-8染色质的关键DNA元件的功能，目标如下：1.测定不同组织和发育阶段的61C7-8开放结构域结构(组蛋白修饰和绝缘蛋白结合)。2.DNA元件对61C7-8结构域结构和转录的影响。测定BEAF结合元件和启动子元件对61C7-8结构域三维折叠的影响。BEAF调控的果蝇活性染色体结构域的定位和初步鉴定以61C7-8为模型，本实验将有助于理解开放的染色质结构域在发育过程中是如何形成的。它将进一步提供关于基本结构域结构的稳定性以及它在不同组织中与基因表达相关的修改程度的信息。这些实验将显示绝缘蛋白BEAF、CP190和Chriz对于染色质修饰的形成和开放染色质结构域的折叠的重要性。此外，他们将解决Chriz和CP190如何独立于BEAF结合而对这两个方面做出贡献，并将解决因子结合和转录活性的相互依赖。结构域中单个BEAF结合位点的消除将使我们能够了解结合在单个位点的因子之间的相互作用，以及可能的结构域内相互作用的层次。最后，实验旨在将我们的61C7-8相关研究扩展到进一步开放的具有类似BEAF连接结构的染色质结构域。更多开放结构域的映射将影响我们目前关于多线和二倍体染色质结构域的概念，特别是我们关于带间开放染色质结构域的概念。在未来，为61C7-8区域建立的方法可能被用于并扩展到依赖于其他绝缘蛋白的开放染色质结构域的研究。

Our current data show that the 61C7-8 locus in salivary glands is formed by a ~20 kb open chromatin domain that contains four protein coding genes and one noncoding transcript. By site directed recombination we generated a wild type and partial deleted versions of the locus at the same ectopic position that were initially characterized by cytogenetic methods. In the proposed project we want to study the function of key DNA-elements for the formation of 61C7-8 chromatin at high resolution with the following objectives:1. Determination of the 61C7-8 open domain structure (histone modification and insulator protein binding) in different tissues and stages of development. 2. Determination of the influence of DNA elements on a 61C7-8 domain structure and transcription.3. Determination of the influence of BEAF-binding- and promoter-elements on the 3D folding of the 61C7-8 domain.4. Mapping and initial characterization of BEAF regulated active chromosomal domains in Drosophila.Taking 61C7-8 as a model the experiments will provide understanding on how an open chromatin domain is formed in development. It will further provide information on the stability of basic domain structure and the extent of its modifications in different tissues in correlation to gene expression. The experiments will show the importance of the insulator proteins BEAF, CP190 and Chriz for the formation of chromatin modifications and folding of an open chromatin domain. Moreover, they will address how Chriz and CP190 may contribute to both aspects independently of BEAF binding and will address the mutual dependency of factor binding and transcriptional activity. The elimination of single BEAF binding sites within the domain will allow us to understand the mutual interaction between factors bound at individual sites and possibly the hierarchy of interactions within the domain.Finally, the experiments are intended to extend our 61C7-8 related studies to further open chromatin domains with a similar BEAF connected architecture. Mapping of more open domains will influence our current concepts on polytene and diploid chromatin domain structure, in particular our concepts on interbands as open chromatin domains. In the future the methods established for the 61C7-8 region may be used and extended for the study of open chromatin domains that depend on other insulator proteins.